title: Dynamic conformational changes in munc18 prevent syntaxin binding.
abstract: The Sec1/munc18 protein family is essential for vesicle fusion in eukaryotic cells via binding to SNARE proteins. Protein kinase C modulates these interactions by phosphorylating munc18a thereby reducing its affinity to one of the central SNARE members, syntaxin-1a. The established hypothesis is that the reduced affinity of the phosphorylated munc18a to syntaxin-1a is a result of local electrostatic repulsion between the two proteins, which interferes with their compatibility. The current study challenges this paradigm and offers a novel mechanistic explanation by revealing a syntaxin-non-binding conformation of munc18a that is induced by the phosphomimetic mutations. In the present study, using molecular dynamics simulations, we explored the dynamics of the wild-type munc18a versus phosphomimetic mutant munc18a. We focused on the structural changes that occur in the cavity between domains 3a and 1, which serves as the main syntaxin-binding site. The results of the simulations suggest that the free wild-type munc18a exhibits a dynamic equilibrium between several conformations differing in the size of its cavity (the main syntaxin-binding site). The flexibility of the cavity's size might facilitate the binding or unbinding of syntaxin. In silico insertion of phosphomimetic mutations into the munc18a structure induces the formation of a conformation where the syntaxin-binding area is rigid and blocked as a result of interactions between residues located on both sides of the cavity. Therefore, we suggest that the reduced affinity of the phosphomimetic mutant/phosphorylated munc18a is a result of the closed-cavity conformation, which makes syntaxin binding energetically and sterically unfavorable. The current study demonstrates the potential of phosphorylation, an essential biological process, to serve as a driving force for dramatic conformational changes of proteins modulating their affinity to target proteins.
authors.affiliation: Department of Neurobiology, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Dana
authors.initials: D
authors.lastname: Bar-On
authors.forename: Esther
authors.initials: E
authors.lastname: Nachliel
authors.forename: Menachem
authors.initials: M
authors.lastname: Gutman
authors.forename: Uri
authors.initials: U
authors.lastname: Ashery
mesh_terms: D000818:Animals; D001665:Binding Sites; D016000:Cluster Analysis; D019295:Computational Biology; D049832:Decapodiformes; D005656:Fungal Proteins / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D051938:Munc18 Proteins / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D046328:Porcupines; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D055672:Static Electricity
chemical_list: D005656:Fungal Proteins; D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins